Method of preventing proliferation of retinal pigment epithelium by retinoic acid receptor agonists

ABSTRACT

Proliferation of retinal pigment epithelium following surgery or trauma or resulting in ocular diseases associated with choroidal neovascularization, such as age related macular degeneration and histoplasmosis syndrome, is prevented by contacting retinal pigment epithelium cells with a therapeutic amount of a retinoic acid receptor (RAR agonist, preferably one with specific activity for retinoic acid receptors. Preferably the RAR agonist is also a potent antagonist of AP1-dependent gene expression. Alternatively, the proliferation of retinal pigment epithelium is ameliorated with a therapeutic amount of an AP-1 antagonist, alone or in combination with an RAR agonist. The drug can be administered by bolus injection into the vitreous cavity using a dosage from about 50 to 150 μg. Or by slow release from liposomes or an oil tamponade injected into the vitreous cavity. Formulations for preventing proliferation of retinal pigment epithelium are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 09/536,221, filedMar. 27, 2000 now U.S. Pat. No. 6,372,753; which is a continuation ofU.S. Ser. No. 08/875,665, filed Jan. 23, 1998 (U.S. Pat. No. 6,075,032,issued Jun. 13, 2000); which is a PCT National Phase Application ofPCT/US96/01505, filed Jan. 31, 1996, which published in English as WO96/23498 on Aug. 8, 1996; and continuation-in-part of U.S. Ser. No.08/383,741, filed Feb. 1, 1995 (U.S. Pat. No. 5,824,685, issued Oct. 20,1998).

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to pharmacological uses of retinoids. Moreparticularly, this invention relates to use of retinoids in treatment ofocular disorders.

2. Description of Related Art

The retinal pigment epithelium (RPE) forms a monolayer of cells beneaththe sensory retina that is normally mitotically inactive except when itis participating in retinal wound repair, in which it plays a centralrole. When wound repair is complete, the RPE usually stopsproliferating; failure to do so can result in blinding disorders such asproliferative vitreoretinopathy (PVR) and disciform scarring. Forinstance, after detachment of the sensory retina, the RPE changes inmorphology and begins to proliferate. Multilayered colonies ofdedifferentiated RPE cells are formed. Cells then begin to migrate intothe subretinal space where they engulf rod outer segments. In someinstances cells migrate onto the surface of the retina and formepiretinal membranes. These events have been implicated in thepathogenesis of proliferative vitreoretinopathy, severe scarringoccurring in association with macular degeneration, and poor or delayedrecovery of vision after retinal reattachment.

Age-related macular degeneration (AMD) is the major cause of blindnessin patients over the age of 60 in the United States. Severe loss ofvision in patients with AMD is usually due to the development ofchoroidal neovascularization (CNV). Laser treatment can ablate CNV andhelp to preserve vision in selected cases not involving the center ofthe retina; however, the treatment benefit is often transient due to thehigh rate of recurrent CNV (50% over 3 years and approximately 60% at 5years) (Macular Photocoagulation Study Group, Arch. Ophthalmol. 204:694-701, 1986). In addition, many patients who develop CNV are not goodcandidates for laser therapy because the CNV is too large for lasertreatment, or the location cannot be determined so that the physiciancannot accurately aim the laser.

Despite these important consequences, little is known about the stimuliinvolved in RPE dedifferentiation and loss of density-dependent growthcontrol. However, it is known that cultured human RPE rapidly becomedepleted of retinoids when maintained in media supplemented with fetalbovine serum (FBS) (S. R. Das, et al., Biochem. J., 250:459, 1988).Retinoids have been implicated in cellular differentiation (S.Strickland, et al., Cell, 15:393-403, 1978; T. R. Brietman, et al.,PNAS, 77:2936-2940, 1980; and are normally present in high levels in RPEin vivo. Retinoids play a prominent role in visual transduction andtherefore their recycling is needed for normal visual function. Thisrecycling occurs through an intimate relationship between thephotoreceptors and the RPE. Disruption of this intimate relationshipduring retinal detachment prevents recycling of retinoids and may be onereason for outer segment degeneration and dedifferentiation of the RPE(P. A. Campochiaro, et al., Invest. Opthalmol. Vis. Sci., 32:65-72,1991).

Incubation of cultured RPE cells with all-trans retinoic acid (RA)inhibits cell proliferation and promotes a morphologic appearance likeRPE in situ (P. A. Campochiaro, et al., supra; J. W. Doyle, et al.,Curr. Eye Res. 11:753-765, 1992). All-trans RA and other derivatives ofvitamin A (generally referred to as retinoids) affect the growth anddifferentiation of many cell types (S. Strickland, et al., supra; T. R.Breitman, et al., Proc. Natl. Acad. Sci. USA, 7:2936-2940, 1980).Therefore, retinoic acid or a related molecule may be one of the signalsthat helps to maintain or re-establish quiescence of RPE and other cellsthat participate in PVR.

The biological effects of retinoids are mediated through nuclearreceptors which are ligand-induced trans-acting factors that bind to DNAresponse elements, modulating the transcription of genes containingthose response elements.

These receptors are divided into two major families, retinoic acidreceptors (RARs) and retinoid X receptors (RXRs). For each family thereare three separate gene products constituting three subtypes designatedα, β, and γ (H. G. Stunnenberg, Bio Essays, 15:309-315, 1993).Alternative splicing of mRNA for these subtypes results in differentisoforms and even greater diversity. The level of expression of RAR andRXR subtypes differs from tissue to tissue, and differences in activityof subtypes may provide some tissue specificity of retinoid effects (P.Dollé, et al., Nature, 342:702-705, 1989; J. L. Rees, et al., Biochem.J., 259:917-919, 1989). Retinoid receptors up-regulate gene expressionby binding to the promoter regions of RA-responsive genes astranscriptionally active RAR-RXR heterodimers (S. Nagpal, et al., EMBOJ., 12:2349-2360, 1993) or RXR homodimers (X. Zhang, et al., Nature,358:587-591, 1992); whereas they down-regulate expression of other genesby antagonizing the effect of transcription factors such as AP-1 (M.Pfahl, Endocrine Review, 14:651-658, 1993; Nagpal, et al., J. Biol. Chem270:923-927, 1995). AP-1 components c-Jun and c-Fos are products ofimmediate early genes which are produced in response to the mitogenicsignals (e.g., growth factors and tumor promoters) arriving at the cellmembrane. The c-Jun/c-Fos complex, in turn, activates the expression ofAP-1-responsive genes involved in cell division and cell proliferation(T. Curran and B. R. Franzo, Jr., Cell, 55:395-397, 1988; I. R. Hart, etal., Biochim. Biophys. Acta. 989:65-84, 1989; P. K. Vogt and T. J. Bos,Trends Biochem. Sci., 14:172-175, 1989). On the other hand, retinoidsinhibit cell proliferation and induce differentiation (L. J. Guoas, etal., in The Retinoids: Biology, Chemistry and Medicine, eds. M. B.Sporn, et al., Raven Press Ltd., New York, pp 443-520, 1994). Therefore,in the light of the above mentioned observations, retinoid mediatedantagonism of AP-1-dependent gene expression provides a basis of theiranti-proliferative effects. Another level of regulation is provided bydifferences in ligand-receptor affinities. All trans-RA is theendogenous ligand for RARs, while that for RXRs is believed to be9-cis-RA (R. A. Heyman, et al., Cell, 68:397-406, 1992; A. A. Levin, etal., Nature, 355:359-361, 1992); however, 9-cis-RA can bind to andactivate the RARs as well. 9-cis RA is a stereoisomer of all-trans RAand is generated from all-trans RA in vivo during metabolism (R. A.Heyman, et al., supra).

Proliferative vitreoretinopathy (PVR) is the most common cause offailure following rhegmatogenous retinal detachment surgery. Despitemeticulous surgical membrane removal and the use of tamponades such assilicone oil (SiO), failure occurs in a large number of cases due todifficulty with complete removal and continuous growth of the membranes.To date, the goals of surgery for PVR are to relieve traction by removalof epiretinal membranes and portions of foreshortened retina whennecessary, surround all retinal breaks with retinopexy, and inject gasor silicone oil into the vitreous cavity to provide retinal tamponadefor a sufficiently long, period of time that all retinal breaks aresealed. Using these techniques, retinal reattachment is achievedin-35-45% of eyes with PVR with one operation, and in up to 71% of eyeswith multiple operations. However, with each operation the visualprognosis worsens (Silicone Study Group, Silicone Study Report No. 2.,Arch Ophthalmol, 110:780-792, 1992). The major cause of failure isreproliferation with regrowth of epiretinal membranes resulting intraction and recurrent detachment. Therefore, prevention ofreproliferation is a major goal in the treatment of PVR.

A number of experiments have been reported using differentantiproliferative agents, such as daunomycin, alone or in combinationwith vitreoretinal surgery. Retinoids are lipid soluble, as mostantiproliferative agents are not. All-trans RA dissolved in SiO wastested in a rabbit model of PVR, and produced a significant and lastingreduction in cellular proliferation. At doses of 2 to 20 μg/ml SiO in 3kilogram rabbits no histological evidence of retinal toxicity was found,and the rate of retinal detachment was decreased from 100% in untreatedcontrols to 44.5% in treated rabbit eyes at 8 weeks (J. J. Araiz, etal., Invest. Ophthalmol. Vis. Sci., 34:522-30, 1993). This mode ofretinoid delivery is suitable for patients with advanced PVR in whomsilicone oil is often used, but is not applicable for use in patientswith early or less severe PVR or those patients at high risk for PVRafter retinal reattachment.

The rabbit model has also been used to test sustained delivery of RAfrom microspheres of biodegradable polymer in treatment of PVR (G. G.Giordano, et al., Invest. Ophthalmol. Vis. Sci., 34:2743-2751, 1993).Filling the vitreous cavity with a suspension of biodegradablemicrospheres into which a total of about 100 μg of all-trans RA wasincorporated significantly decreased tractional retinal detachment (TRD)in eyes treated with gas compression vitrectomy and fibroblastinjection. Toxicity was limited to localized areas of inflammation feltto represent a foreign body reaction.

Retinoic acid, its geometric isomer 13-cis-retinoic acid, and syntheticderivatives have numerous biological effects in several tissues.Retinoids are currently used as the first line treatment for acutemyelogenous leukemia (R. P. Warrell, Jr., et al., N. Engl. J. Med.,324:1385-1393, 1991), as adjuvant therapy for several types ofmetastatic carcinomas (K. Dhingra, et al., Invest. New Drugs, 11:39-43,1993; S. M. Lipman, et al., J. Natl. Can. Inst., 85:499-500, 1993), andfor treatment of psoriasis and other skin disorders. While these variedeffects of retinoids provide multiple clinical applications, they arealso the basis for undesired effects and toxicity that can impede thetreatment of any one particular disorder. For instance, 13-cis-retinoicacid is associated with teratogenic effects when administered topregnant females of child-bearing age. Thus, the need exists foradditional synthetic retinoid analogs that avoid these toxic effects foruse in treatment of PVR. Also, understanding the mechanism by whichretinoids exert their antiproliferative effect in RPE cells will enablethe development of new and adjunctive therapeutic agents for PVR andrelated diseases.

SUMMARY OF THE INVENTION

Proliferation of retinal pigment epithelium following surgery or traumaor in ocular diseases associated with choroidal neovascularization, suchas age related macular degeneration and histoplasmosis syndrome, istreated by contacting retinal pigment epithelium cells with atherapeutic amount of a retinoic acid receptor (RAR) agonist, preferablyone with specific activity for retinoic acid receptors and with potentability in inhibiting AP1-dependant gene expression. This proliferationis also treated with therapeutic amounts of other agents that inhibitAP1-dependent activity, used singly or in combination with RAR agonists.The contacting can be accomplished by bolus injection into the vitreouscavity or by providing the RAR agonist in a slow release format, such asencapsulated into liposomes or dissolved in a liquid tamponade injectedinto the vitreous cavity or periocular space. Formulations forpreventing proliferation of retinal pigment epithelium are alsoprovided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing molar concentration-dependent potency ofretinoid agonists for inhibition of serum stimulated DNA synthesis inhuman RPE cells after incubation for 7 days. 745=Compound 745;183=Compound 183; RA=retinoic acid; 9-cis-RA=9-cis-retinoic acid;659=Compound 659; 701=Compound 701.

FIG. 2 is a series of micrographs at magnification×150 using a phasecontrast microscope showing the effect of retinoid agonists on RPE cellmorphology. Magnification×150.

FIGS. 2A-D show cells (4×10⁴) from a 60 year-old donor grown in fourdifferent media. FIG. 2A=serum alone; 2B=5% serum supplemented with 1 μMall-trans retinoic acid; 2C=5% serum supplemented with 1 μm of Compound701; 2D=5% serum supplemented with 1 μm of Compound 183.

FIGS. 2E-H show cells (4×10⁴) from a 76 year-old donor grown in fourdifferent media. FIG. 2A=serum alone; 2B=5% serum supplemented with 1 μMall-trans retinoic acid; 2C=5% serum supplemented with 1 μm of Compound701; 2D=5% serum supplemented with 1 μm of Compound 183.

FIG. 3 is a graph showing the effect of several nuclear receptoragonists alone or in combination with retinoic acid in RPEproliferation. RA=retinoic acid; Dex and Dexa=dexamethasone; T3=thyroidhormone; D3=1,25-dihydroxyvitamin D₃.

A DETAILED DESCRIPTION OF THE INVENTION

Provided herein is a method for treating proliferative vitreoretinopathy(PVR) and other disorders of retinal wound repair by contacting retinalpigmented epithelial (RPE) cells with a therapeutic amount of one ormore compounds having activity as a retinoic acid receptor (RAR)agonist. It has been discovered that RAR agonists prevent theproliferation of RPE cells in vitro by promoting density arrest and adifferentiated morphology in cultured RPE mediated through anRAR-activated pathway, and are also clinically effective for inhibitingtraction retinal detachment in animals, such as humans, as is commonlysuffered following eye surgery and other wounds to the retina that causedetachment of the RPE from the photoreceptors. These results indicatethat inhibition of traction retinal detachment is mediated by aRAR-activated pathway, but not by a RXR-mediated pathway. Thus, RARagonists are generally useful in the treatment of conditions in whichthere is excessive proliferation of RPE cells leading to retinalscarring or retinal detachment.

On the other hand, RXR agonists have no such useful clinical effects.Further, the primary mechanism by which RAR agonists inhibit RPEproliferation is by antagonism of AP1 activity. Thus, RAR agonists withspecific anti-AP1 activity, other agents that block AP1 activity, orcombinations of these other agents and RAR agonists with anti-AP1activity can be used to treat PVR.

It is shown in the Examples herein that the above-described retinoidcompounds of this invention produce a differentiated phenotype inretinal epithelial cells. One of the differentiated functions of retinalpigmented epithelial cells is to prevent growth of new blood vesselsfrom the choroid through Bruch's membrane into the space beneath theRPE, or into the subretinal space. This process is called choroidalneovascularization. It occurs in several disease processes, the mostcommon of which is age-related macular degeneration. Consequently,treatment with the retinoid compounds of this invention helps tomaintain RPE in the differentiated state and, therefore, helps toprevent choroidal neovascularization in patients.

The patients with age-related macular degeneration represent the largestgroup in which choroidal neovascularization is a major problem. Inaddition, younger patients who suffer choroidal neovascularization, suchas those with ocular histoplasmosis syndrome, are also benefited bytreatment with the retinoid compounds of this invention.

In the method of this invention, a therapeutically effective amount ofan RAR agonist having potent activity as an antagonist of AP1 or anotheranti-AP1 agent or a combination of these agents is introduced into thevitreous cavity or periocular space of a patient who has PVR orchoroidal neovascularization, or is at risk of developing theseconditions due to disease, surgery, trauma or aging. In the case ofsurgery, it is particularly preferred that the RAR agonist or otheranti-AP1 agent or combination be given after surgery for PVR to preventreproliferation of RPE cells and redetachment of the retina.

As used herein, the term “a therapeutically effective amount” of an RARagonist is an amount calculated to achieve and maintain a therapeuticlevel in the vitreous cavity, if introduced directly into the vitreouscavity or periocular space, or in the bloodstream, if administeredperipherally over the period of time desired in a human or animal suchas to substantially inhibit proliferation and thus restoredifferentiation of RPE. It is preferred that the therapeutic amount bean amount sufficient to inhibit proliferation of at least 50 percent,more preferably 80 percent of the RPE cells in an eye under treatment.The therapeutic amount will vary with the potency of each RAR agonist,the amount required for the desired therapeutic or other effect, therate of elimination or breakdown of the substance by the body once ithas entered the vitreous cavity or bloodstream, and the amount of theRAR agonist in the formulation. In accordance with conventional prudentformulating practices, a dosage near the lower end of the useful rangeof a particular agent is usually employed initially, and the dosage isincreased or decreased as indicated from the observed response, as inthe routine procedure of the physician.

For administration directly into the vitreous cavity of the eye, anamount in the range between about 50 and 150 μg administered withinabout 24 hours following surgery or trauma generally provides protectionagainst development of PVR. In an alternative embodiment, a seconddosage of the RAR agonist after an interval of several hours, usuallybetween about 8 and 36 hours, preferably about 24 hours, is injectedintravitreally or subconjunctivally. Alternatively, a combination ofintravitreal and subconjunctival injection of the retinoid, eithersimultaneously or at the above described spaced interval, can be used toadminister the retinoid. For intravitreal injection, it is preferredthat the RAR agonist be injected into the anterior vitreous cavity usingtopical or retrobulbar anesthesia. In an alternative embodiment, the RARagonist is introduced intravitreally using a drug delivery vehicle. Forinstance, the RAR agonist can be dissolved in a biologically inert fluidthat is also useful as a mechanical tamponade to help keep the retina inplace, preferably an oil such as silicone oil in which the retinoid issoluble. However, for RAR agonists having partial miscibility, a liquidother than an oil can be used.

However, intravitreous injection of the retinoids as a bolus injectioncan result in focal areas of retinal damage. In addition, it has beendiscovered that the therapeutic effects of the retinoids of thisinvention are delayed in onset and reversible. Therefore, it isadvantageous to administer the retinoids utilizing a method of a slowrelease, for instance by intravitreal injection of the dose of retinoidencapsulated in a microvesicle, such as a liposome, from which the doseis released over the course of several days, preferably between about 3to 20 days. Alternatively, the drug can be formulated for slow release,such as incorporation into a slow release polymer from which the dosageof drug is slowly released over the course of several days, for examplefrom 2 to 30 days.

As is known in the art, liposomes are generally derived fromphospholipids or other lipid substances. Liposomes are formed by mono-or multilamellar hydrated liquid crystals that are dispersed in anaqueous medium. Any non-toxic, physiologically acceptable andmetabolizable lipid capable of forming liposomes can be used. Thepresent compositions in liposome form can contain stabilizers,preservatives, excipients, and the like in addition to the agent. Thepreferred lipids are the phospholipids and the phosphatidyl cholines(lecithins), both natural and synthetic.

Methods to form liposomes are known in the art. See, for example,Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, NewYork, N.Y. (1976), p. 33 et seq.

Regardless of the mode of administration, the RAR agonist can be eithernaturally occurring or a synthetic retinoid, preferably having selectiveactivity as an agonist for RARs and high potency in antagonism ofAP-1-dependent gene expression. Examples of naturally occurringretinoids with activity as RAR agonists are all-trans retinoic acid(all-trans RA) and 9-cis retinoic acid (9-cis RA), which arestereoisomers, all-trans RA being naturally converted into 9-cis RAduring metabolism (J. G. Allen, et al., Pharmac. Ther., 40:1-27, 1989).However, 9-cis RA has activity as agonists for both RARs and RXRs, andall-trans-RA, because of its metabolism or chemical isomerization to9-cis-RA, can also effectively activate both RARs and RXRs; whereas thepreferred retinoid compounds for use in the practice of this inventionhave specific activity as RAR agonists.

Synthetically prepared retinoids are well known in the art. For example,U.S. Pat. No. 5,234,926, which is incorporated herein by reference inits entirety, discloses methods of synthesizing disubstituted acetylenesbearing heteroaromatic and heterobicyclic groups with selective activityas RAR agonists. U.S. Pat. No. 4,326,055, which is incorporated hereinby reference in its entirety, discloses methods for synthesizing5,6,7,8-tetrahydro naphthyl and indanyl stilbene derivatives withretinoid-like activity. Since it is known that proliferation of culturedRPE cells is inhibited in vitro by retinoid compounds having activity asRAR agonists and not by compounds having activity as RXR agonists,anti-proliferative retinoid compounds can readily be selected bydetermining whether they have RAR activity, for instance by utilizingwell known in vitro transacivation assay techniques such as thatdisclosed by M. Pfahl, et al., Methods in Enzymology, 1:256-270, 1990,and as illustrated in Example 1 of this invention. A RAR selectiveagonist will prevent cell overgrowth, resulting in a cell morphologyindistinguishable from that caused by all-trans RA.

Examples of synthetic RAR agonists suitable for use, in the practice ofthis invention are ethyl6-[2-(4,4-dimethylthiochroman-6-yl)ethynyl]nicotinate (Compound 168) and6-[2-(4,4-dimethylchroman-6-yl)ethynyl]nicotinic acid (Compound 299),whose synthesis is disclosed in U.S. Pat. No. 5,234,926 as Examples 6and 24, respectively; andp-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)propenyl]-benzoicacid (Compound 183), whose synthesis is disclosed in U.S. Pat. No.4,326,055. By contrast, an example of an RXR selective agonist is2-[(E)-2-(5, 6, 7, 8-tetrahydro-3, 5, 5, 8,8-pentamethylnaphthaleen-2-yl)propen-1-yl]thiophene-4-carboxylic acid(Compound 701), whose synthesis is disclosed in U.S. Pat. No. 5,324,840,Example 11.

Preferably the RAR agonist is selected to be metabolically stable and toremain completely specific for the RAR pathway. Thus, all-trans RA,which isomerized to 9-cis RA during metabolism and results in activationof both the RAR and RXR pathways, is not preferred for use in thepractice of this invention.

The cross-talk between the retinioid and AP-1 signal transductionpathways can be manipulated for therapeutic benefit inhyperproliferative diseases. This has been demonstrated in thisinvention since the retinoids that are STRONG antagonists ofAP-1-dependent gene expression, are also STRONG INHIBITORS of RPE cellproliferation. Potent anti-proliferative RAR-agonists can be screened bydetermining their ability to inhibit AP-1-dependent gene expression in atransient transfection CAT assay as illustrated in Example 2 of thisinvention.

The following examples illustrate the manner in which the invention canbe practiced. It is understood, however, that the examples are for thepurpose of illustration and the invention is not to be regarded aslimited to any of the specific materials or conditions therein.

EXAMPLE 1

The transactivation properties of retinoids were determined by measuringtheir ability to induce transcription in the HeLa cells by transientlycotransfecting them with a receptor gene construct and a reporter gene.Since retinoid receptors are members of the steroid receptor family ofnuclear receptors that are characterized by homologous functionaldomains, hybrid receptors were used that contain the amino terminus andDNA-binding domain of the estrogen receptor and the hormone-bindingdomain of the retinoid receptors, either RAR-α, β, γ, or RXR-α. TheseER/RAR receptors activate transcription by binding to promoter sequencesrecognized by the estrogen receptor (estrogen response element), but doso in response to a retinoid (D. Benbrook, et al., Nature, 333:669-672,1988). Previous studies have shown that the activation characteristicsof hybrid receptors are determined by their ligand binding domain. Todetermine activation of the hybrid constructs by the retinoids, anestrogen receptor-responsive reporter gene was used that cannot beactivated by endogenous retinoid receptors, which are present in most,if not all, mammalian cells.

The Cationic Liposome Mediated Transfection Assay by which the activityof a test compound as, a potential agonist of the RXR and RAR receptorsites is determined, is performed substantially as reported by P. L.Feigner, et al., Focus, 11:2, 1989, which is incorporated herein byreference, and is described below first in principle and thereafter inthe form of specific instructions how to perform the assay.

In connection with this assay it is known that retinoic acid receptorsare a member of the steroid/thyroid receptor super family and that theycontain domains which are interchangeable within individual receptors.Thus, plasmids for chimeric retinoid receptors containing estrogen DNAbinding domain and estrogen response element chloramphenicolacetyl-transferase (CAT) enzyme are constructed and are grown inspecific cultured bacteria. These plasmids respectively code forchimeric RAR_(α), RAR_(β), RAR_(γ), or RXR_(α) receptor proteins, andfor the chloramphenicol acetyl A transferase (CAT) enzyme protein. Thebacteria with these plasmids are obtainable in accordance with theprocedure set forth in the article titled “Nuclear Retinoic AcidReceptors: Cloning, Analysis, and Function”, M. Pfahl, et al., Methodsin Enzymology, 189: 256-270, 1990), which is incorporated herein byreference. The detailed procedure for isolating the DNA plasmids fromthe respective bacteria is also set forth below in detail, in the formof specific instructions under the title “Supercoiled PlasmidIsolation”.

Thus, in accordance with the test procedure, a DNA plasmid that codesfor one of the chimeric RAR_(α), RAR_(β), RAR_(γ), or RXR_(α) receptorproteins is transfected into cultures of HeLa cells. It is for thispurpose that HeLa cells are grown in a medium during the first day ofthe assay detailed below as the “Cationic Liposome Mediated TransfectionAssay”. In the transfection procedure, which is performed during thesecond day of the transfection assay, the DNA plasmid coding for the CATenzyme is also added to each cell culture, in addition to the respectivechimeric RAR_(α), RAR_(β), RAR_(γ), or RXR_(α) coding plasmid.

As is known and will be readily understood by those skilled in the art,especially in view of the above-cited M. Pfahl, et al, article, chimericretinoid receptors involved in this assay include a ligand bindingdomain that recognizes and binds specific agonist molecules, such asretinoic acid and analogs. These chimeric protein receptors (which wereconstructed in accordance with the teachings of the M. Pfahl, et al.article) also contain a DNA binding domain, which is capable of bindingto the “estrogen response element” (a DNA fragment) attached to the DNAplasmid coding for the CAT enzyme. The nature of the interaction is suchthat only when an agonist (such as retinoic acid or analog) is bound tothe ligand binding domain of the respective RAR_(α), RAR_(β), RAR_(γ),or RXR_(α) receptor, is the receptor bound through its DNA-bindingdomain to the estrogen response element of theestrogen-response-element-chloramphenicol-acetyl, transferase-construct(ERE-CAT) and capable of initiating transcription of messenger RNA forthe CAT enzyme. In other words, through multiple interactions CAT enzymeis manufactured by the HeLa cell in this assay only if an appropriateagonist ligand binds to the ligand binding site of the respectiveretinoid receptor.

The estrogen response-element-chloramphenicol acetyl-transferaseconstruct (ERE-CAT) is itself obtained in accordance with the proceduredescribed in G. U. Ryssel, et al. (Cell, 46:1053-1061, 1986), which isincorporated herein by reference. This procedure per se is well known inthe art. The specific detailed procedure for isolating and obtaining theestrogen-response-element chloramphenicol-acetyl-transferase-construct(ERE-CAT) from bacteria is described in the procedure titled“Supercoiled Plasmid Isolation”.

In addition to the foregoing, lipofectin (LF) is also added to each cellculture. The purpose of the lipofectin is to facilitate transport ofplasmids through the cell membrane. The lipofectin used in the procedureis available commercially.

As will be well understood by those skilled in the art, as a result oftransfection with the 25 respective DNA plasmids coding for RAR_(α),RAR_(β), RAR_(γ), or RXR_(α) chimeric receptors and as a result oftransfection with the ERA-CAT (which codes for the CAT enzyme asdescribed above), the aforementioned plasmids are incorporated into theHeLa cells cultured in the assay. The retinoid receptor plasmids undergotranscription (into m-RNA) and subsequent translation into thecorresponding chimeric receptor protein. Therefore, the HeLa cellscultures obtained in this manner manufacture the respective RAR_(α),RAR_(β, RAR) _(γ), or RXR_(α) chimeric receptor protein. As a result oftransfection with the ERA-CAT the cell cultures of this assay alsocontain the genetic information for manufacturing the CAT enzyme.However, as is noted above, the latter genetic information is nottranscribed, and the CAT enzyme is not manufactured by the respectivecell cultures of this assay, unless an appropriate agonist compoundbinds to and activates the respective RAR_(α), RAR_(β), RAR_(γ), orRXR_(α) chimeric receptor protein in the cell and this activatedagonist-receptor complex binds to the estrogen response element of theERE-CAT construct.

The assay procedure is continued by adding, on the third day of theassay, an appropriate reference compound and the test compound (agonistor prospective agonist) to the respective HeLa cell culture, preferablyin varying concentrations. As a result of this addition, if the testcompound is an agonist, it binds to the respective RAR_(α), RAR_(β),RAR_(γ), or RXR_(α) chimeric receptor protein, and consequently thegenetic information which codes for the CAT enzyme is transcribed in thecell, whereby CAT enzyme is made by the cell.

After lysis of the cell, which is performed on the fourth day of thebelow-detailed assay procedure, the activity of CAT enzyme in aliquotportions of the lysate is measured. This is done by incubating thelysate with chloramphenicol and tritium labeled acetyl coenzyme A. As afinal measurement, the amount of tritium labeled acetyl chloramphenicol,which is formed in the enzymatic reaction involving the CAT enzyme, ismeasured in a scintillation counter.

The reference compound is retinoic acid (all trans) for the assaysinvolving the RAR_(α), RAR_(β), and RAR_(γ), receptors, and4-(E)-2-(5,6,7,8-tetrahydro3,5,5,8,8-pentamethyl-naphthalen-2-yl)-propen-1-yl benzoic acid (alsodesignated Compound 440 in this application) for the RXR_(α) chimericreceptor. The data obtained in the assay are evaluated and expressed asfollows. For each test compound and for each subspecies of the RARreceptors, a graph (or the mathematical equivalent of a graph) isprepared where the “counts per minute” (cpm) obtained in thescintillation counter measurements are plotted (on the y axis) againstthe concentration of the test compound. A similar graph (or mathematicalequivalent) is prepared for retinoic acid. EC50 of the test compound isdefined as that concentration of the test compound which providesone-half (50%) of the maximum cpm number (maximum CAT enzyme activity)obtained in the same receptor in the same assay with the referencecompound retinoic acid.

To evaluate and express the data obtained in the assay for the RXR_(α)the same type of graph (or mathematical-equivalent) is prepared for thetest compound, and also for the reference compound, Compound 440. Thisreference compound is a known agonist of the RXR_(α) receptor site. EC₅₀is that concentration of the test compound which gives one half (50%) ofthe counts per minute (CAT enzyme activity) of the maximum cpm obtainedwith Compound 440 on the same receptor in the same assay.

SUPERCOILED PLASMID ISOLATION Large Scale 1L Prep

DNA Isolation

1. Place cells on ice for 15 minutes. Harvest the bacterial cells (E.coli) by spinning down in 250 ml nalgene tubes at 7 k rpm, 10 minutes at4° C. using JA14 rotor, Beckman J2-21 M centrifuge. Discard thesupernatant.

2. To each cell pellet add 1.0 ml Solution I, vortex to resuspend thepellet. Transfer the 1.0 ml of cells from one bottle to another.Transfer this to a 50 ml Oakridge tube. Use 4 ml of Solution I and washthe bottles again transferring from one bottle to the next. Transferthis also into the Oakridge tube. Using a pipet bring up the totalvolume to 16 ml with Solution I and mix the solution. Transfer 8 ml to asecond Oak ridge tube. Store at room temperature for 5 minutes.

Solution I

50 mM glucose, 25 mM Tris-Cl pH 8, 10 mM EDNA pH 8

3. Add to each tube ml of freshly prepared Solution II. Mix contentsgently by inverting the tube several times. Store on ice for 10 minutes.After this time the liquid should be clear with no aggregates. (If thereare clumps, then the cells were not resuspended well enough previously.)

Solution II

1% sodium dodecylsulfate (SDS), 0.2N NaOH (4 ml 10% SDS, 0.8 ml 10NNaOH, 35.2 ml water)

4. Add 12 ml, (or as much as will fit) of ice-cold Solution III. Mix thecontents of tube by inverting it sharply several times. A whiteflocculent precipitate should appear. Store on ice for 10 minutes.

Solution III

Prepare as follows: to 60 ml 5M potassium acetate, add 11.5 ml ofglacial acetic acid and 28.5 ml water

5. Centrifuge at 4° C. in a Beckman J2-21M centrifuge JA20 rotor,(Beckman Instruments, Carlsbad, Calif.) at 17 k rpm for 30 minutes.

6. Pipet approximately 12 ml of supernatant from the Oakridge tubes into6 baked Corex tubes. Add 0.6 volumes of isopropanol (7.2 ml) mix byinversion and store at room temperature for 15 minutes to precipitateDNA.

7. Warm Beckman centrifuge by spinning JA20 rotor at 14 k rpm for 15minutes at 20° C.

8. Pellet DNA at 20° C. in the J2-21M centrifuge, JA20 rotor at 10.5 krpm for 30 minutes (use adapters for corex tubes).

9. Pour off supernatant, dry inside of tube with pasteur pipet on avacuum flask.

10. Dry in vacuum dessicator for 10 minutes (longer drying time willmake it hard to dissolve pellet).

Purify plasmid DNA by centrifugation to equilibrium in CsCl densitygradients.

11. Dissolve pellet by adding 1 ml TE (10 mM Tris-Cl pH 8, 1 mM EDNApH8) to each Corex tube. Place tubes in 37° C. water bath to helppellets to dissolve faster (15-30 minutes).

12. Transfer liquid from like batch into one tube. Bring volume to 8.5ml with TE.

13. Add 100 μl RNase, DNase free (2U/μl, source Boehringer MannheimBiochemical (BMB) (Indianapolis, Ind.).

14. Add 400 μl of 10 mg/ml Ethidium Bromide.

15. Add 9.0 g CsCl and mix using a pasteur pipet.

16. Add solution to two 13×51 mm Beckman polyallomer quick-sealcentrifuge tubes.

17. Spin at 50 k rpm for 12 hours in Beckman ultracentrifuge, VTi65.2rotor, 20° C.

18. After ultracentrifugation, two bands of DNA should be visible. Theupper band consists of linear bacterial DNA and nicked circular plasmidDNA. The lower band consists of closed circular plasmid DNA. Only thelower CsCl-banded DNA is removed from the tube with a 3-ml syringefitted to an 21-gauge needle (Needle is inserted into the side of thetube and 1.5-2 ml is removed).

19. Preparation for Second CsCl centrifugation:

(9 ml-vol 1st CsCl band)-number g CsCl

(9 ml-vol 1st band-100 μl 10 mg/ml Ethidium

Bromide-50 μl RNase)-ml TE pH 8.0

Combine 1st band, TE, CsCl, RNase and EtBr.

20. Add solution to 2 quick-seal tubes.

21. Spin at 50 k for 12 hours or 60 k rpm for 4 hours inultracentrifuge, VTi65.2 rotor, 20° C.

22. Remove twice CsCl-banded DNA (lower band only) to a 5 ml Falcon snaptube (as in step 18).

Extraction of ethidium bromide

23. Under fumehood add an equal volume isoamyl alcohol, vortex, spin atroom temperature at 1500 rpm in Beckman TJ-6 centrifuge for 3 minutes.

24. Transfer bottom aqueous layer to fresh tube. Repeat 3-4 times oruntil aqueous layer is clear (no pink color).

25. Transfer clear aqueous layer to Spectra/Por 3 dialysis tubing, mwco3500. (Tie a knot in the bottom of tubing before clamping dialysistubing.) Add liquid using a pasteur pipet. Clamp top or dialysis tubing.Using a rubber band suspend tubing in 2.8 L TE (28 ml 1M Tris-Cl, pH8,5.6 ml 0.500M EDNA, pH8). Always handle dialysis tubing carefully, withgloves.

26. Dialyze aqueous phase against several changes of 2.8 L TE pH8 (1×2-4hours, overnight and 1×2-4 hours the next day).

27. In the tissue culture hood transfer the dialyzed DNA, into sterilemicrocentrifuge tubes. Label tubes and store at −20° C.

Cationic Liposome-Mediated Transfection

Reference: P. L. Felgner, et al., Focus, 11: 2, 1989.

Use Sterile Technique Throughout

Grow up HeLa or CV-1 cells in T-125 culture flask. Cells are passedtwice a week, usually on Monday and Friday (0.5 ml cells into 15 mlmedium).

DAY 1: Plating Cells

1. Trypsinize and collect cells from T-162 cm² culture flask. Countcells using a hemocytometer. Usually, this amount of cells is enough forsixteen 12-well plates.

2. Based on the cell number, dilute cells in medium (D-MEM low glucose,10% fetal bovine serum (FBS), 2 mM 20 Glu) to a concentration of 60,000cells per well.

Example Cell Calculation:

want 40,000 cells/well and 200 wells

have (X) cells/ml

therefore, 40,000 cells/well×200 wells-total # ml cells

(X) cells/ml

needed

Using a 200 ml filter unit receiver add total, #ml cells to medium andbring final volume to 200 ml. Mix well by pipetting.

3. Add 1.0 ml of cells per well using a sterile 12.5 ml dropper. Shakeplates back and forth (do not swirl). Incubate at 37° C. in a humidified5% CO₂ environment overnight. Cells are about 40% confluent prior totransfection.

Transfection: DAY 2 Preparation DNA/Lipofectin Complex

1. Using 50 ml polystyrene tubes prepare Lipofectin (LF) and DNAseparately. Determine vol of LF and DNA needed for 2 μg LF/well, 500 ngERE-CAT DNA/well, 100 ng ER/RAR DNA per well. Determine total volumeneeded for experiment. (DNA concentration will vary between each plasmidprep). Separately dilute LF and DNA in Opti-Mem media (Gibco-BRL;Gaithersburg, Md.) a volume of 25 ul×#wells: Vol Opti-Mem

1=(25 ul×#wells)−total vol. DNA or LF.

2. Add the diluted LF to the diluted DNA and swirl tube gently. Let sitat room temperature for 10 min.

3. Aspirate off the medium from the wells and wash 2× using 0.5 mlOpti-Mem I (sterile 12.5 ml combitip, setting 2).

4. Add the DNA/LF complex to vol of Opti-Mem: (450 μl×# wells). Inverttube to mix. Using a sterile 12.5 ml dropper add 500 μl to each well.Shake plates back and forth to mix, do not swirl.

5. Incubate the cells for 6 hours at 37° C. in a humidified 5% CO₂incubator.

6. After 6 hours add 0.5 ml medium to each well (Dulbecco's ModifiedEagle's Media(D-MEM) low glucose, 20% FBS charcoal treated, 2 mM Glu)Use 12.5 dropper and place back in the incubator.

10 DAY 3: Drug Addition

1. 18 hours after the start of transfection add retinoids in triplicate(10 μl) using a sterile 0.5 ml dropper and incubate for 20-24 hours at37° C. in a humidified 5% CO₂ environment.

Drug Dilutions

weight (g)×1×100 ml=______ ml

Acetone

mol. wt (g/mol) 0.005 mol/L L

EXAMPLE

Retinoids are dissolved in acetone to a concentration of 5 mM andfurther diluted to 1 mM in EtOH. If retinoids do not go into solution,place tube in hot water for 5 seconds followed by vigorous vortexing.Each experiment may have a different dilution scheme. For 2concentrations per order of magnitude use a 3.16-fold dilution asfollows: To labeled sterile 12×75 mm tubes add 1080 μl of 100% EtOH.Using the 1 mM solution, transfer 500 μl to the next tube (316 μM).Vortex and repeat the transfer to the next tube down the line. Someretinoids are light sensitive, especially RA and 13-cis RA, and shouldbe used with a red or very dim light.

EXAMPLE

Drug Dilution Vol add Final: 5 mM (initial) to well −log [conc.] 1 mM 105.0 316 μM 10 5.5 100 μM 10 6.0 31.6 μM 10 6.5 10 μM 10 7.0 3.16 μM 107.5 1 μM 10 8.0 316 nM 10 8.5 100 nM 10 9.0 31.6 nM 10 9.5 10 nM 10 10.03.16 nM 10 10.5 1.0 nM 10 11.0

Day 4 Mixed Phase Cat Assay

1. Wash cells in 12 mm wells once with 0.50 ml 1×PBS (no Ca/Mg).

2. Using a 5 ml pipet add 100 μl of a ice cold 1% Triton, 1 mM Tris-ClpH7.8, 2 mM EDTA pH8, DNase I. Prepared as follows:

Lysis Buffer (Hypotonic Buffer)

2.0 mg DNase I (Sigma, St. Louis, Mo.)

4.925 ml water

50.0 μl 100% Triton X-100 (BMB)

5.0 μl 1M Tris-Cl pH 7.8

20.0 μl 0.5M EDTA pH 8

5.0 ml

3. Place on ice for 60 minutes. Agitate occasionally.

4. Transfer 50 μl of lysate from 3 wells at a time using titertrekmultichannel pipet with tips attached to channels #1, #3, #6 to 96U-bottom well (Costar, Cambridge, Mass.). Place (unused lysate) platesat −20° C.

5. Using a 1.25 ml combipipet (setting 1) add 50 μl premix per well,gently shake plates and incubate 37° C. for 2 hours.

Vol. per Vol. per Blank vol. reaction × _(———) (# assays) = total 47.0 27.0 μl buffer I (250 mM Tris-Cl pH 7.8, 5 mM EDTA 1.5 1.5 μl 1 mM HCl*** 20.0 μl 5 mM Chloramphenicol (make fresh in buffer I)  0.75 0.75 μl4 mM Acetyl CoA in water (make fresh)  0.80 0.80 μl 3H-Acetyl CoA (NewEngland Nuclear, Boston, MA # NET-290L, 200 mCi/mmol

6. Using a titertrek multichannel pipet, add 100 μl of 7M Urea(Mallincrokt, Chesterfield, Md.) into each reaction well to quench thereaction. Do six at a time.

7. Using a titertrek multichannel pipet, transfer 200 μl reactionmixture into a 5 ml plastic scintillation vial (Research ProductsInternational #125514, Mount Prospect, Ill.). Do three reactions at atime.

8. Add 1 ml 0.80% 2,5 Diphenyloxazole(PPO)/toluene (3.2 g PPO(Mallinckrodt-RPI)/4L Toluene (Mallinckrodt ScintillAR™). Vortexvigorously for 5 seconds and allow the phases to separate for 15minutes. Count cpm, for 2.0 min-Beckman LS 3801.

The retinoid activities were determined at receptor subtypes RAR-α,RAR-β, RAR-γ and RXR-α.

Table 1 below shows the EC₅₀ for activation of RAR-α, β, γ, and RXR-αfor each of the retinoids used in this study. Four of the agents showedgood selectivity for activation of RAR receptors compared to RXRreceptors. Two agents, 9-cis-RA and synthetic agonist Compound 659activate both RAR and RXR receptors.

TABLE 1 POTENCY OF RETINOID AGONISTS AT INDUCING TRANCRIPITIONALACTIVATION THROUGH SPECIFIC RECEPTORS EC₅₀ (nm) Compound No. RAR_(α)RAR_(β) RAR_(γ) RXR_(α) 183 30  2  1 15,000 745 45 235 590 N/A All transRA  5    1.5    0.5 >10,000   9-cis RA 100   3  5    20 659 N/A  20 100   15 701 N/A 3000  1000    100

Example 2

Primary cultures of human retinal pigment epithelium (RPE) cells wereestablished from eyes obtained from the Old Dominion Eye Bank (Richmond,Va.) or the Eye Bank of Maryland (Baltimore, Md.) using the techniquedescribed in P. A. Campochiaro, et al., Invest. Ophthalmol. Vis. Sci.,27:1615-1621, 1986 which is incorporated herein by reference in itsentirety.

The RPE cell lines used in this study were from two donors aged 60 and76 years, respectively, and each was stained uniformly for cytokeratinsusing a known technique (K. H. Leschey, et al., Invest. Ophthalmol. Vis.Sci., 31:839-846, 1990). All-trans RA was obtained from Sigma (St.Louis, Mo.) and 9-cis retinoic acid and synthetic retinoid agonists wereobtained from Allergan, Inc. (Irvine, Calif.).

For [3H]thymidine incorporation, RPE cells at passage 3 or 4 werelightly trypsinized and plated in 16-mm wells of 24-well plates. Thetransfected cells were allowed to attach overnight and then the mediacontaining 5% fetal bovine serum (FBS) were supplemented with retinoidsor vehicle alone. Stock solutions (10⁻³M) of retinoids were prepared indimethylsulfoxide (DMSO) and stored as frozen aliquots until used; thehighest final concentration of DMSO was 0.1% and was also used forcontrol cultures. The media containing freshly prepared retinoids werechanged every three days. At 7 or 10 days 2 μCi/ml of [³H]thymidine(specific activity, 6.7 Ci/mM; New England Nuclear, Boston, Mass.) wasadded to the cultures and incorporation was measured as previouslydescribed (Leschey, supra).

The potency of retinoid agonists for inhibition of serum stimulated DNAsynthesis in human RPE cells was tested. RPE cells (4×10⁴ cells) wereplated in 16-mm wells of 24-well plates and grown for 6 days in DMEMcontaining 10% fetal bovine serum (Life Technologies, Inc.) supplementedwith various concentrations of one of the following compounds: Compound#745, 183, 659, or 701, all-trans retinoic acid (RA) or 9-cis retinoicacid (9-cis-RA). The cells were shifted to serum-free media containingthe same concentration of retinoid and then pulsed with 10% serum for 18hours, after which [³H]thymidine incorporation was measured. Thymidineincorporation for cells grown in control medium was used to calculatethe percent inhibition of thymidine incorporation induced by fourconcentrations of each retinoid (each concentration tested intriplicate) which were then used to generate each line.

As shown by the results summarized in FIG. 1, incubation of RPE cellsfor 7 days in all-trans-RA or 9-cis-RA results in dose-dependentinhibition of [³H]thymidine incorporation.

As shown by the results summarized in Table 2, a 10 day incubationresults in greater inhibition and the potencies of all-trans RA and9-cis-RA are very similar. Incubation of RPE cells for 7 or 10 days ineach of four synthetic retinoids that selectively activate RAR receptorsresults in strong inhibition of [³H]thymidine incorporation.

TABLE 2 EFFECT OF RETINOID TREATMENT FOR TEN DAYS ON [³H] THYMIDINEINCORPORATION IN RPE CELLS Percent Inhibition Compound No. 5 × 10⁻⁸ M10⁻⁷ M 10⁻⁶ M 183 76.7 98.6 98.8 745 79.2 98.0 98.6 All trans RA 52.678.3 99.4 9-cis RA 57.8 79.8 98.2 659 20.3 73.2 98.7 701 12.0 48.8 74.6

The RXR receptor-selective agonist (Compound 701) was a much lesseffective inhibitor of RPE [³H]thymidine incorporation than theRAR-selective agonists, and agonists that activate both RXR and RARreceptors (9-cis RA and Compound 659) were not better inhibitors thanthose that activate only RAR receptors.

Example 3

The anti-AP-1 properties of retinoids are determined by measuring theirability to inhibit AP-1-dependent gene expression in HeLa cells bytransiently cotransfecting them with a reporter gene and a receptorexpression vector. Since the DNA binding domain of the RARs is involvedin the inhibition of AP-1-dependent gene expression (R. Schule, et al.,Proc. Natl. Acad. Sci. USA, 88:6092-6096, 1991), holoreceptors of RARs(α, β, USA, 88:6092-6096, 1991), holoreceptors of RARs (α, β, and γ) areused in transfection assays to quantitate the relative potency ofretinoids in antagonism of AP-1-dependent gene expression.

Recombinant Plasmids

The expression vectors for RARs (α, β, and γ) have been described (E. A.Allegretto, et al., J. Biol. Chem., 268:26625-26633, 1993).AP-1-reporter plasmid construct Str-AP-1-CAT was prepared by cloning −84to +1 base pairs of rat stromelysin-1 promoter (L. M. Matrisian, et al.,6:1679-1686, 1986) in Hind III-Bam HI sites of pBLCAT3 (B. Luckow and G.Schutz, Nucl. Acids Res., 15:5490, 1987). This sequence of stromelysin-1promoter contains an AP-1 motif as its sole enhancer element (R. C.Nicholson, et al., EMBO J., 9:4443-4454, 1990). The promoter sequencewas prepared by annealing two synthetic oligonucleotides 5′-AGAAGCTT ATGGAA GCA ATT ATG AGT CAG TTT GCG GGT GAC TCT GCA AAT ACT GCC ACT CTA TAAAAG TTG GGC TCA GAA AGG TGG ACC TCG A GGATCCAG-3′ (SEQ ID NO:1) AND5′-CT GGATCC TCG AGG TCC ACC TTT CTG AGC CCA ACT TTT ATA GAG TGG CAG TATTTG CAG AGT CAC CCG CAA ACT GAC TCA TAA TTG CTT CCA T AAGCTT CT-3′ (SEQID NO:2) containing Hind III and Bam HI restriction sites at their ends.The specific details for obtaining the supercoiled plasmid expressionand reporter vectors have been described in detail in Example 1.

Transfection of Cells an CAT Assays

For retinoid-mediated AP-1-antagonism assay, HeLa cells grown inDulbecco's modified Eagle's medium (DMEM), containing 10% fetal bovineserum (FBS, Life Technologies, Inc.) are transfected using the cationicliposome-mediated transfection procedure (P. L. Feigner, et al., Focus,11:2, 1989). Cells are plated 18 h before transfection at about 40%confluence (40,000-50,000 cells/well) in a 12-well tissue culture plate(Costar, Cambrigde, Mass.). Cells are transfected with 1 μg of reporterconstruct Str-AP-1-CAT and 0.2 μg of human RAR α, β, or γ expressionvectors, along with 2 μg of Lipofectamine (Life Technologies, Inc.) foreach well in a total volume of 500 μl. The details of plating ofrecombinant HeLa cells have been described in Example 1.DNA/Lipofectamine complexes obtained by mixing 2 μg Lipofectamine/well,1 μg Str-AP-1-CAT/well, and 0.2 μg RAR expression vector in a 50 mlpolystyrene tube are treated and incubated with HeLa cells in exactlythe same manner as described for DNA/Lipofectin complexes in Example 1of this invention. DNA is precipitated with Lipofectamine for 30 min atroom temperature before transfer to cells. Five hours post-transfection,500 μl of DMEM containing 20% charcoal treated FBS (Gemini Bioproducts,Inc., Calif.) is added. All the transfections are performed intriplicate. Test retinoids (at 10⁻¹⁰ to 10⁻⁷ M concentrations) are added18 h post-transfection and 6 h later the cells are treated with12-0-tetradecanoyl phorbyl-14-acetate (TPA) to induce AP-1 activity.

Retinoids are dissolved in acetone to a concentration of 5 mM andfurther diluted from this stock solution using ethanol. The next dayafter washing with phosphate buffered saline without calcium andmagnesium (Life Technologies, Inc.), the cells are harvested and lysedfor 60 min with occasional agitation using a hypotonic buffer (100μl/well) containing Dnase I, Triton X-100, Tris-HCl and EDTA asdescribed in the mixed phase CAT assay section of Example 1 of thisinvention. CAT activity is assayed in 50 μl of the lysed cell extractusing [³H]acetyl CoA (DuPont NEN) in a 96-well U-bottom plate (Costar).The CAT activity is quantified by counting the amount of ³H-acetylatedforms of chloramphenicol using a liquid scintillation counter. Thedetailed procedures of CAT assay and scintillation counting of labeledacetylated forms of chloramphenical are described in the Example 1 ofthis invention

TABLE 3 AP1 INHIBITION AND RPE CELL GROWTH INHIBITION AP1 InhibitionCompound RPE 148 EC₅₀ (nM)^(b) Number Structure IC₅₀ (nM)^(a) RARα RARβRARγ All-trans RA <10      0.07    0.23   <0.01  13-cis RA 30    0.08 10 1 521 10    0.01   0.4    0.01 121 30   0.6  5   1.9 509 30 22 NA   0.01 183 <10      0 17   <0.01     0.53 659 10  >10²   14 22 867 >10³    >10²    >10²    >10²   870  >10³    >10²    >10²    >10²   NA =Not Available ^(a)Retinoid (nM) required for 50% inhibition of RLPE cellgrowth relative to mock treated RPE cells. ^(b)Retinoid (nM) requiredfor 50% inhibition of Str-AP1-CAT activity in transient transfectionassays.

The effects of retinoid agonists in inhibiting AP1 activity as describedin Example 3 are shown in Table 3. Also shown in Table 3 are the effectsof the same retinoids in inhibiting RPE cell proliferation measured asdescribed in Example 2. Compounds that are potent inhibitors of AP1activity (e.g., compounds 521, 183 and 659) are all effective inhibitorsof RPE cell proliferation. In contrast, compounds that are ineffectiveinhibitors of AP1 activity (e.g., compounds 867 and 810) are alsoineffective in inhibiting RPE cell proliferation.

Example 4

Other nuclear receptors, including the thyroid hormone receptor, whichis activated by thyroid hormone (T3), the glucocorticoid receptor, whichis activated by dexamethasone (Dex), and the vitamin D receptor, whichis activated by 1,25-dihydroxyvitamin D₃ (D3), can mediate anti-AP1activity. The effect of dexamethasone, T3 and D3 on RPE cellproliferation (measured as described in Example 2) was examined aloneand in combination with retinoic acid (RA). The results are shown inFIG. 3. Dexamethasone inhibited RPE cell proliferation, but T3 and D3did not. However, each of these agents had a synergistic effect whenused in combination with retinoic acid. This provides supportiveevidence that the mechanism by which RAR agonists inhibit RPE cellproliferation is by antagonism of AP1 activity and suggest that othereffective inhibitors of AP1 activity, when used alone or in combinationwith RAR agonists, will be useful for the treatment of PVR.

Example 5

To determine the effect of various retinoids on RPE cell morphology, RPEcells from the two donors described in Example 1 were plated in 35-mmwells and grown on plastic for 10 days in media containing 5% serum withor without one of three retinoids (1 μm); all-trans RA; a RAR selectiveagonist (Compound 183); or a RXR selective agonist (Compound 701).

As shown in FIGS. 2A and 2B, the RPE cells grown in 5% serum containingmedia without other additions showed extensive overgrowth with numerousprocesses from one cell extending over neighboring cells. Cells treatedwith all-trans RA did not exhibit cell overgrowth, resulting in amorphology more like RPE in situ as shown in FIGS. 2C. and 2D. The RARselective agonist also prevented cell overgrowth, resulting in amorphology indistinguishable from that caused by all-trans RA (FIGS. 2Eand 2F); whereas the RXR-selective agonist failed to prevent cellovergrowth, resulting in a morphology indistinguishable from thecontrols, as shown in FIGS. 2G and 2H. Trypan blue exclusion showedstaining of less than 10% of cells in all cultures supplemented witheach of the retinoids and was not statistically different from controls.

Example 6

The effect of intravitreous injections of retinoids was investigated inthe rabbit cell injection model of PVR first described by H. A. Sen, etal. (Arch. Ophthalmol., 106:1291-1294, 1988), which is incorporatedherein by reference in its entirety. Briefly, pigmented rabbits wereanesthetized with a subcutaneous injection of 5 mg/kg of xylazine and 25mg/kg of ketamine, and the pupil of one eye was dilated with 2½%phenylephrine. Under direct observation 5×10⁵ RPE cells were injectedinto the vitreous cavity just anterior to the optic nerve. In initialexperiments intravitreous injections of 100 μg of all-trans-RA, aselective RXR agonist (Compound 701), a selective RAR agonist (Compound183), or vehicle were administered to the rabbits.

TABLE 4 EFFECT OF INTRAVITREOUS INJECTION OF RETINOIDS ON THE FREQUENCYAND SEVERITY OF TRACTION RETINAL DETACHMENT IN THE RABBIT CELL INJECTIONMODEL OF PVR 14 Days 28 Days Partial Total Partial Total Total Any N RDRD RD RD RD (%) RD (%) Vehicle Control* 8 2 5 1 6 75 87.5    VehicleControl 5 3 2 1 4 80 100       All-trans-RA 100 μg* 9 2 0 4 0 0 44.4+ All-trans-RA 100 μg 7 1 0 1 1 14.3 28.6+  All-trans-RA 50 μg 6 0 4 0 466.7 66.7    RXR Agonist 8 3 3 2 5 62.5 87.5    (Compound 701) 100 μg*RXR Agonist 5 2 2 1 3 60 80      (Compound 701) 100 μg RAR Agonist 5 0 00 0 0   0++   (Compound 183) 100 μg* RAR Agonist 9 1 2 1 2 22 33+  (Compound 183) 50 μg *Two Injections; one on day 1 after cell injectionand one on day 2 +p <0.05 for difference from control by Student'spaired t-test ++p <0.01 for difference from control by Student's pairedt-test

Rabbits were examined by indirect ophthalmoscopy on days 7, 14 and 28and traction retinal detachments were graded. After injection of alltrans-RA, a localized yellow cloud of precipitate appeared in thevitreous and remained for several weeks, and injection of the otheragonists resulted in a while cloud. The localized clouds decreased insize and cleared over 2 to 3 weeks. There were no visible changes in theretina. As shown by the data summarized in Table 4, eyes injected withall-trans RA had fewer (p(0.05) and less severe traction retinaldetachments than eyes injected with vehicle alone, while eyes injectedwith the RXR-selective agonist did not differ from controls.

In the initial group of rabbits, five received two intravitreousinjections of the RAR-selective agonist. None of these rabbits developedretinal detachments, but all experienced hair loss and loss of appetite,and two rabbits died before completion of the 28 day observation period.It was felt that the hair loss and possibly the loss of appetite anddeath were due to systemic toxicity from drug that had gotten out of theeye. Another group of rabbits was tested in which a single intravitreousinjection of 50 μg of the RAR agonist, 50 μg and 100 μg of RA, or 100 μgof RXR agonist (701), or vehicle alone was given one day after RPE cellinjection. The eyes injected with the RAR agonist or with RA had fewer(p(0.05) and less severe retinal detachments than the control eyes, butthe eyes injected with the RXR agonist were not statistically differentfrom controls. None of the rabbits experienced hair loss and none died;their appetites appeared to be normal.

Example 7

To examine for ocular toxicity, rabbits were given a singleintravitreous injection of 100 μg of all-trans-RA, the RAR-selectiveagonist (compound 183), the RXR-selective agonist, (compound 701), anagonsit that activates both RAR and RXR (compound 659), or vehiclealone. The rabbits were killed two weeks after injection. The results ofhistopathologic examination of the retinas of the rabbits are summarizedin Table 5. In general, the retinas were well-preserved and showed onlymild changes believed to represent no more than artifact. As shown bythe data in Table 5 below, all of the eyes, including those injectedwith vehicle, showed mild vitritis. One of the eyes injected with theRXR agonist and one injected with the agonist showing activity with bothRXR and RAR receptors exhibited a few focal areas of retinal necrosis.The same RXR-injected eye also showed a few focal areas of inner retinaledema, and similar focal areas of inner edema were seen in one eyeinjected with the RAR agonist. Occasional focal areas of photoreceptordegeneration were seen in one eye injected with the RAR agonist and inone eye injected with the agonist having both RXR and RAR activity.

TABLE 6 EFFECT OF INTRAVITREAL INJECTION OF THE RAR AGONISTS 168 AND 299ON THE DEVELOPMENT OF TRACTION RETINAL DETACHMENT IN RABBITS AFTERINJECTION OF HUMAN RPE CELLS Compound RD at 7 Days RD at 14 Days RD at28 Days % Total % Any No. N Partial Total Partial Total Partial Total RDRD Control 12 0 0 0 0 6 0 0 50 168 12 1 0 1 0 7 1 8 80 299 10 0 0 1 1 44 40 80 RA 2 0 0 0 0 2 0 0 100

Areas of retinal whitening were noted in several of the drug-injectedeyes, and were associated, with drug precipitates, suggesting thatlocalized areas of high concentration of Compound 168 or 299 might causeretinal damage that could be responsible for the high rate of retinaldetachment. It was concluded that the poor solubility of retinoids inthe vitreous resulted in precipitates and localized areas of highconcentration that resulted in toxicity.

Example 9

Due to the toxicity of certain RAR agonists and because the effects ofretinoids are delayed in onset and reversible, methods for sustainedrelease of the retinoids were investigated. To explore alternativemethods of sustained release delivery, rabbits were givensubconjunctival injections of 0.3 mg of Compound 168, 299, or RA once aday for five days after intravitreous injection of 5×10⁵ human RPEcells. The results as shown in Table 7 demonstrate that, as comparedwith the control, subconjunctival injections of all three retinoidsadministered over a course of five days decreased TRD. The therapeuticeffect of the RAR specific agonists was greater than that of RA, whichis an agonist for both RXRs and RARs. There were no signs of anytoxicity to the retina.

TABLE 7 EFFECT OF SUBCONJUNCTIVAL INJECTION OF THE RAR AGONISTS 168 AND299 ON THE DEVELOPMENT OF TRACTION RETINAL DETACHMENT IN RABBITS AFTERINJECTION OF HUMAN RPE CELLS Compound RD at 7 Days RD at 14 Days RD at28 Days % Total % Any No. N Partial Total Partial Total Partial Total RDRD Control 12 0 0 0 0 6 0 0 50 168 6 0 0 0 0 1 0 16 16 299 4 0 0 0 0 0 00 0 RA 4 0 0 0 0 0 0 0 0

Example 10

The effect of subconjunctival injection of RAR agonists was tested in arabbit model of PVR as described in Example 7, except that human dermalfibroblasts were injected into the vitreous cavity in the place of RPEcells. Generally, this model tends to have a higher rate of retinaldetachment and more severe detachments than does the model utilizing RPEcells. However, as shown by the data in Table 8, subconjunctivalinjections of Compounds 168, 299, and all-trans RA all decreased thenumber of retinal detachments and slowed the rate at which theydeveloped as compared with the results of the tests described in Example9, although the improvement over the control was not as great as inExample 9.

TABLE 8 EFFECT OF SUBCONJUNCTIVAL INJECTION OF THE RAR AGONISTS 168 AND299 ON THE DEVELOPMENT OF TRACTION RETINAL DETACHMENT IN RABBITS AFTERINJECTION OF HUMAN DERMAL FIBROBLASTS Compound RD at 7 Days RD at 14Days RD at 28 Days % Total % Any No. N Partial Total Partial TotalPartial Total RD RD Control 10 2 0 5 0 7 1 10 80 168 10 0 0 0 0 4 2 2060 299 10 0 0 0 0 4 0 0 40 RA 10 0 0 0 0 5 0 0 50

Example 11

In this experiment the retinoid was incorporated into lipidmicrovesicles utilizing standard techniques. Microvesicles encapsulatingall-trans RA, Compounds 168 and 299, and vehicle were injected into thevitreous cavity of rabbits on the day after intravitreous injection of5×10⁵ human dermal fibroblasts. As shown by the data in Table 9 below,microvesicles containing Compound 168 were very effective in preventingtraction retinal detachment, while those containing Compound 299 orall-trans RA were not effective as compared with the control group.

TABLE 9 EFFECT OF INTRAVITREOUS INJECTION OF LIPID MICROVESICLESCONTAINING 168 OR 299 ON THE DEVELOPMENT OF TRACTION RETINAL DETACHMENTIN RABBITS AFTER INJECTION OF HUMAN DERMAL FIBROBLASTS Compound RD at 7Days RD at 14 Days RD at 28 Days % Total % Any No. N Partial TotalPartial Total Partial Total RD RD Control 11 2 0 5 1 7 2 18 82 168 7 0 00 0 0 2 29 29 299 3 0 0 0 0 0 3 100 100 RA 6 1 0 1 1 0 4 67 67

Retinoids were incorporated into lipid microvesicles and injectedsubconjunctivally into the eyes of rabbits on the day afterintravitreous injection of 5×10⁵ human dermal fibroblasts. As shown bythe data in Table 10 below, Compound 168 was substantially moreeffective at inhibiting tractional retinal detachment than were theother retinoids or vehicle.

TABLE 10 EFFECT OF SUBCONJUNCTIVAL INJECTION OF LIPID MICROVESICLESCONTAINING 168 OR 299 ON THE DEVELOPMENT OF TRACTION RETINAL DETACHMENTIN RABBITS AFTER INJECTION OF HUMAN DERMAL FIBROBLASTS Compound RD at 7Days RD at 14 Days RD at 28 Days % Total % Any No. N Partial TotalPartial Total Partial Total RD RD Control 2 0 0 1 0 0 2 100 100 168 8 00 1 0 0 2 33 33 299 8 0 0 0 1 1 4 50 63 RA 8 0 0 0 1 4 1 13 63

The foregoing description of the invention is exemplary for purposes ofillustration and explanation. It should be understood that variousmodifications can be made without departing from the spirit and scope ofthe invention. Accordingly, the following claims are intended to beinterpreted to embrace all such modifications.

SUMMARY OF SEQUENCES

SEQ ID NO: 1 is an oligonucleotide primer for the stromelysin-1promoter.

SEQ ID NO: 2 is an oligonucleotide primer for the stromelysin-1promoter.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: THE JOHNS HOPKINS UNIVERSITY SCHOOL OF MEDICINE

(ii) TITLE OF INVENTION: METHOD OF PREVENTING PROLIFERATION OF RETINALPIGMENT EPITHELIUM BY RETINOIC ACID RECEPTOR AGONISTS

(iii) NUMBER OF SEQUENCES: 2

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Fish & Richardson P.C.

(B) STREET: 4225 Executive Square, Suite 1400

(C) CITY: La Jolla

(D) STATE: California

(E) COUNTRY: USA

(F) ZIP: 92037

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/Ms-DOS

(D) SOFTWARE: PatentIn Release #1.0, Version 1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: PCT/US95/

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Learn, June M.

(B) REGISTRATION NUMBER: 31,238

(C) REFERENCE/DOCKET) NUMBER: 07265/023 WO1

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE; (619) 670-5070

(B) TELEFAX: (619) 679-5099

(2) INFORMATION FOR SEQ ED NO:1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 101 Base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: COS

(B) LOCATION: 1 . . . 101

(xi) SEQUENCE DESCRIPTION: SEQ ED NO:1:

AGAAGCTTAT GGAAGCAATT ATGAGTCAGT TTGCGGGTGA CTCTGCAAAT ACTGCCACTC  60

TATAAAAGTT GGGCTCAGAA AGGTGGACCT CGAGGATCCA G  101

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 101 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY:

(B) LOCATION: 1 . . . 101

(xi) SEQUENCE DESCRIPTION: SEQ ED NO:2:

CTGGATCCTC GAGGTCCACC TTTCTGAGCC CAACTTTTAT AGAGTGGCAG TATTTGCAGA  60

GTCACCCGCA AACTGACTCA TAATTGCTTC CATAAGCTTC T  101

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 2 <210> SEQ ID NO 1 <211> LENGTH: 101<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetically generated olig #onucleotide<400> SEQUENCE: 1agaagcttat ggaagcaatt atgagtcagt ttgcgggtga ctctgcaaat ac#tgccactc     60 tataaaagtt gggctcagaa aggtggacct cgaggatcca g    #                   #  101 <210> SEQ ID NO 2 <211> LENGTH: 101<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetically generated olig #onucleotide<400> SEQUENCE: 2ctggatcctc gaggtccacc tttctgagcc caacttttat agagtggcag ta#tttgcaga     60 gtcacccgca aactgactca taattgcttc cataagcttc t    #                   #  101

What is claimed is:
 1. A method for amelioration of traction retinaldetachment comprising contacting the retinal pigmented epithelial cellsof a subject in need thereof with a therapeutic amount of a retinoicacid receptor agonist.